1. Field of the Invention
The present invention is in the field of plant genetics, and relates to a method for identifying resistance of a rice plant to rice blast. In particular, the present invention relates to a method for identifying field resistance of a rice plant to rice blast by determining a genotype of the genome of a rice plant using a DNA marker which is closely linked to a gene controlling the field resistance to rice blast.
2. Description of the Related Art
Usually, when a plant is infected with a pathogen such as a virus, the plant is inhibited from growing, resulting in eventual death. On the other hand, it has been known that certain plants exhibit resistance to a pathogen. It is believed that such plants have a pathogen resistance gene. Several such resistance genes have been confirmed to exist. However, the ability of a pathogen resistance gene in a plant is generally limited to the particular combination of the plant and the specific pathogen, and a gene expressing resistance to one pathogen may not express a trait of resisting any pathogen.
In the field of breeding and improving of plants, a plant having a pathogen resistance gene or an insect resistance gene has been crossed with another plant which does not have such a pathogen or insect resistance gene but has another desirable trait so as to generate a number of improved varieties into which a genetic trait of disease resistance has been introduced. In view of preventing an ecological pollution and ensuring safety for human bodies, it is believed that the generation of such resistant varieties through crossing will become more and more important in the future as a biological epidemic prevention method which utilizes the organism's innate function and does not require agrochemicals.
Rice blast is a plant disease caused by infection with Pyricularia oryzae, and is the most prominent disease for rice. Once a rice plant is infected with this fungus, the disease will generally prevail across the farm to completely destroy all the rice plants in the farm. Thus, rice blast is a serious problem for rice breeders. One way to address this problem is to create a variety resistant to rice blast by conferring the resistance to the rice through cross breeding.
The rice blast resistance gene of a rice plant was first found in Japan (Sasaki, R. Jpn. J. Genetics 1:81–85 (1922)). Thereafter, many genes have been confirmed to be related to the rice blast resistance. By crossing a variety having a rice blast resistance gene (i.e., a rice blast resistant variety) with a variety lacking a rice blast resistance gene (i.e., a rice blast non-resistant variety), it is possible to introduce a new trait of rice blast resistance to a variety which is not resistant to rice blast but has other desirable traits (e.g., early maturity, tolerance to cold injuries, good grain and eating qualities).
In conventional breeding, a field testing method has been employed for testing whether a resistance gene of one variety has been introduced to hybrid progeny lines crossed with another variety. According to this method, an individual to be tested is inoculated with Pyricularia oryzas, and the rice blast resistance thereof is identified based on the degree of susceptibility or resistance. However, such a testing method requires: breeding an extremely large number of individuals to be tested: inoculating the individuals with the fungus; breeding the inoculated individuals: and evaluating the degree of susceptibility or resistance to rice blast for each of the individuals to be tested. The series of steps requires substantial labor and time on the order of several months, and is not suitable for quick and efficient development of resistant varieties. Moreover, such a testing method needs to be carried out within a special facility in order to avoid propagation of rice blast to other neighboring farms.
In recent years, various molecular biological techniques have been developed, allowing for organisms, tissues or cells to be tested for the presence of a specific gene for determining the genotype thereof. Thus, when the relationship between a gene and the corresponding trait, i.e., phenotype is specified, the phenotype can be indirectly determined by confirming the presence of the gene.
Exemplary techniques for use in such a genotype analysis include a restriction fragment length polymorphism (RFLP) analysis and a random amplified polymorphic DNAs (RAPD) analysis. The RFLF analysis is based on the difference in the number and length (polymorphism) of the restriction fragments which are obtained by processing a genomic DNA with a restriction enzyme which only acts upon a particular base sequence site. Specifically, when a genomic DNA, which is the principal element of the gene, is digested with a restriction enzyme, many DNA fragments of various lengths are obtained. Different individuals or lines often have very similar numbers and lengths of restriction fragments. Typically, however, various mutations exist in the DNA for each individual, whereby slight differences may be observed in the number and length of the restriction fragments. Such differences are called the “restriction fragment length polymorphism”. By comparing the differences, the genotype can be determined. The analysis based on the polymorphism requires a useful DNA marker which is closely linked to a gene locus responsible for a genotype of interest. Therefore, it is necessary to identify such a useful DNA marker in order to improve the reliability of the results of the genotype analysis.
The resistance of a rice plant to rice blast is generally classified into two categories: a true resistance; and a field resistance. The true resistance is controlled by a single gene, which exhibits resistance to rice blast on its own. However, as the blast fungus survives, it may mutate and acquire infectivity to a rice plant which used to exhibit a resistance thereto. Therefore, the true resistance may be suddenly lost. In fact, a case has been known where a resistant rice variety cultivated in a farm was infected with rice blast only after 2 years from the time when the farm started growing the variety. In contrast, a field resistance is controlled by a plurality of, probably several, genes. Thus, even when a part of the resistance controlled by one of the genes is lost, for example, the rice variety can be prevented from being seriously damaged by rice blast by virtue of the remaining resistance controlled by the other genes.
As described above, since the true resistance is controlled by a single gene, the heredity thereof is simple and easy to study. A recent report shows that Pi-b, a resistance gene belonging to the true resistance category, has been isolated (Japanese Laid-Open Publication No. 7-163371). While the field resistance is practically highly valuable, it is relatively more difficult to study than the true resistance because the heredity thereof is complicated and the determination of the degree of resistance is likely to be influenced by environmental conditions. Therefore, the field resistance has not been studied as much as the true resistance.
It has been reported that a number of different gene loci related to the field resistance to rice blast were identified by means of the polymorphic analysis using a conventional gene marker (Goto, I., Ann. Phytophatol. Coc. Jpn. 36:304–312 (1970); Higashi, T. and Saito, S. Japan J. Breed. 35:438–448 (1985); Shinoda, H. et al., Bull. Chugoku Nat. Agric. Exp. Static. A20:1–25 (1971)). However, to the knowledge of the present inventors, no report has shown, that a useful DNA marker has been identified which is closely linked to the gene controlling the field resistance.
Thus, there has been a demand for specifying the gene locus which is related to the field resistance to rice blast so as to identify a DNA marker which is closely linked to the gene controlling the resistance. There has also been a demand for developing a method for conveniently and accurately identifying the genotype for the field resistance using such a DNA marker. Such a method would also be useful for efficiently breeding a resistant variety.